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Human adipocytes stimulate invasion of breast cancer MCF-7 cells by secreting IGFBP-2.

Wang C, Gao C, Meng K, Qiao H, Wang Y - PLoS ONE (2015)

Bottom Line: In addition, MMP-2 was remarkably up-regulated, whereas E-cadherin was down-regulated in these MCF-7 cells.We found that IGFBP-2 enhanced the invasion ability of MCF-7 cells in vitro more prominently than did the other factors.In vivo, metastatic human breast tumors had higher levels of MMP-2 than did non-metastatic tumor tissue, whereas adipocytes around metastatic breast tumors had higher levels of IGFBP-2 than did adipocytes surrounding non-metastatic breast tumors.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Chemistry for Life Science & Jiangsu Key Laboratory of Molecular Medicine, Medical School of Nanjing University, Nanjing 210093, China.

ABSTRACT

Background and aims: A better understanding of the effects of human adipocytes on breast cancer cells may lead to the development of new treatment strategies. We explored the effects of adipocytes on the migration and invasion of breast cancer cells both in vitro and in vivo.

Methods: To study the reciprocal effects of adipocytes and cancer cells, we co-cultured human mature adipocytes and breast cancer cells in a system devoid of heterogeneous cell-cell contact. To analyze the factors that were secreted from adipocytes and that affected the invasive abilities of breast cancer cells, we detected different cytokines in various co-culture media. To study the communication of mature adipocytes and breast cancer cells in vivo, we chose 10 metastatic pathologic samples and 10 non-metastatic pathologic samples to do immunostaining.

Results: The co-culture media of human MCF-7 breast cancer cells and human mature adipocytes increased motility of MCF-7 cells. In addition, MMP-2 was remarkably up-regulated, whereas E-cadherin was down-regulated in these MCF-7 cells. Based on our co-culture medium chip results, we chose four candidate cytokines and tested their influence on metastasis individually. We found that IGFBP-2 enhanced the invasion ability of MCF-7 cells in vitro more prominently than did the other factors. In vivo, metastatic human breast tumors had higher levels of MMP-2 than did non-metastatic tumor tissue, whereas adipocytes around metastatic breast tumors had higher levels of IGFBP-2 than did adipocytes surrounding non-metastatic breast tumors.

Conclusions: IGFBP-2 secreted by mature adipocytes plays a key role in promoting the metastatic ability of MCF-7 breast cancer cells.

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Function of the medium generated from the co-culture of MCF-7 cells and mature adipocytes.1A. MCF-7 cells were stimulated with different conditioned media for 24 hours, and then mitosis was inhibited by incubation with mitomycin C for 30 minutes. C-CM and A-CM pre-treated MCF-7 cells showed no change in the scratch width, whereas CAA-CM pre-treated cells had 26%±1.5% wound healing and many cells were found scattered in the middle of the scratch (100 × magnification). 1B. MCF-7 cells treated with CAA-CM showed greater invasion ability than A-CM and C-CM. Quantification was performed by dissolving crystal violet with 10% acetic acid and detecting the OD values with a microplate reader at 595 nm. Pictures were taken at 200 × magnification. The data shown are the means of triplicate wells, and the error bars represent the S.D (**: P<0.01). 1C. Quantification of E-cadherin, N-cadherin, β-catenin, ZO-1, Vimentin, MMP-2 and MMP-9 in MCF-7 cells treated with different media. These mRNAs were detected by quantitative RT-PCR and normalized to GAPDH. The primers used for quantitative PCR are listed in Table 1. The ratios of signal were analyzed for statistical significance using the t-test (*: P<0.05, ***: P<0.001).
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pone.0119348.g001: Function of the medium generated from the co-culture of MCF-7 cells and mature adipocytes.1A. MCF-7 cells were stimulated with different conditioned media for 24 hours, and then mitosis was inhibited by incubation with mitomycin C for 30 minutes. C-CM and A-CM pre-treated MCF-7 cells showed no change in the scratch width, whereas CAA-CM pre-treated cells had 26%±1.5% wound healing and many cells were found scattered in the middle of the scratch (100 × magnification). 1B. MCF-7 cells treated with CAA-CM showed greater invasion ability than A-CM and C-CM. Quantification was performed by dissolving crystal violet with 10% acetic acid and detecting the OD values with a microplate reader at 595 nm. Pictures were taken at 200 × magnification. The data shown are the means of triplicate wells, and the error bars represent the S.D (**: P<0.01). 1C. Quantification of E-cadherin, N-cadherin, β-catenin, ZO-1, Vimentin, MMP-2 and MMP-9 in MCF-7 cells treated with different media. These mRNAs were detected by quantitative RT-PCR and normalized to GAPDH. The primers used for quantitative PCR are listed in Table 1. The ratios of signal were analyzed for statistical significance using the t-test (*: P<0.05, ***: P<0.001).

Mentions: To address the effect of adipocyte-secreted factors on the breast cancer cells, we used conditioned media from 2-day-old homotypic cultures or co-cultures to stimulate MCF-7 cells that had not been previously exposed to any conditioned media. We used MCF-7, which is a cancer cell line with low metastatic potential, for scratch assays and found that medium conditioned by co-cultures (CAA-CM) significantly accelerated the closure of the cell-cleared area compared to unconditioned control medium (C-CM) (Fig. 1A). In invasion assays, CAA-CM caused a significant increase in the number of invasive MCF-7 cells, whereas cells treated with A-CM or control medium showed little invasion (Fig. 1B). This observation suggested that co-cultures might exhibit emergent properties that were not present in the homotypic cultures. To determine the mechanism of advanced migration and invasion ability in breast cancer cells, we chose to assess the mRNA expression levels of several metastasis-associated genes. MMP-2 was remarkably up-regulated whereas E-cadherin was down-regulated in MCF-7 cells treated with CAA-CM compared to those treated with C-CM (Fig. 1C). Taken together, these data suggest that the co-culture of breast cancer cells with mature adipocytes might cause the secretion of some soluble factor that increases breast cancer cell migration and invasion.


Human adipocytes stimulate invasion of breast cancer MCF-7 cells by secreting IGFBP-2.

Wang C, Gao C, Meng K, Qiao H, Wang Y - PLoS ONE (2015)

Function of the medium generated from the co-culture of MCF-7 cells and mature adipocytes.1A. MCF-7 cells were stimulated with different conditioned media for 24 hours, and then mitosis was inhibited by incubation with mitomycin C for 30 minutes. C-CM and A-CM pre-treated MCF-7 cells showed no change in the scratch width, whereas CAA-CM pre-treated cells had 26%±1.5% wound healing and many cells were found scattered in the middle of the scratch (100 × magnification). 1B. MCF-7 cells treated with CAA-CM showed greater invasion ability than A-CM and C-CM. Quantification was performed by dissolving crystal violet with 10% acetic acid and detecting the OD values with a microplate reader at 595 nm. Pictures were taken at 200 × magnification. The data shown are the means of triplicate wells, and the error bars represent the S.D (**: P<0.01). 1C. Quantification of E-cadherin, N-cadherin, β-catenin, ZO-1, Vimentin, MMP-2 and MMP-9 in MCF-7 cells treated with different media. These mRNAs were detected by quantitative RT-PCR and normalized to GAPDH. The primers used for quantitative PCR are listed in Table 1. The ratios of signal were analyzed for statistical significance using the t-test (*: P<0.05, ***: P<0.001).
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pone.0119348.g001: Function of the medium generated from the co-culture of MCF-7 cells and mature adipocytes.1A. MCF-7 cells were stimulated with different conditioned media for 24 hours, and then mitosis was inhibited by incubation with mitomycin C for 30 minutes. C-CM and A-CM pre-treated MCF-7 cells showed no change in the scratch width, whereas CAA-CM pre-treated cells had 26%±1.5% wound healing and many cells were found scattered in the middle of the scratch (100 × magnification). 1B. MCF-7 cells treated with CAA-CM showed greater invasion ability than A-CM and C-CM. Quantification was performed by dissolving crystal violet with 10% acetic acid and detecting the OD values with a microplate reader at 595 nm. Pictures were taken at 200 × magnification. The data shown are the means of triplicate wells, and the error bars represent the S.D (**: P<0.01). 1C. Quantification of E-cadherin, N-cadherin, β-catenin, ZO-1, Vimentin, MMP-2 and MMP-9 in MCF-7 cells treated with different media. These mRNAs were detected by quantitative RT-PCR and normalized to GAPDH. The primers used for quantitative PCR are listed in Table 1. The ratios of signal were analyzed for statistical significance using the t-test (*: P<0.05, ***: P<0.001).
Mentions: To address the effect of adipocyte-secreted factors on the breast cancer cells, we used conditioned media from 2-day-old homotypic cultures or co-cultures to stimulate MCF-7 cells that had not been previously exposed to any conditioned media. We used MCF-7, which is a cancer cell line with low metastatic potential, for scratch assays and found that medium conditioned by co-cultures (CAA-CM) significantly accelerated the closure of the cell-cleared area compared to unconditioned control medium (C-CM) (Fig. 1A). In invasion assays, CAA-CM caused a significant increase in the number of invasive MCF-7 cells, whereas cells treated with A-CM or control medium showed little invasion (Fig. 1B). This observation suggested that co-cultures might exhibit emergent properties that were not present in the homotypic cultures. To determine the mechanism of advanced migration and invasion ability in breast cancer cells, we chose to assess the mRNA expression levels of several metastasis-associated genes. MMP-2 was remarkably up-regulated whereas E-cadherin was down-regulated in MCF-7 cells treated with CAA-CM compared to those treated with C-CM (Fig. 1C). Taken together, these data suggest that the co-culture of breast cancer cells with mature adipocytes might cause the secretion of some soluble factor that increases breast cancer cell migration and invasion.

Bottom Line: In addition, MMP-2 was remarkably up-regulated, whereas E-cadherin was down-regulated in these MCF-7 cells.We found that IGFBP-2 enhanced the invasion ability of MCF-7 cells in vitro more prominently than did the other factors.In vivo, metastatic human breast tumors had higher levels of MMP-2 than did non-metastatic tumor tissue, whereas adipocytes around metastatic breast tumors had higher levels of IGFBP-2 than did adipocytes surrounding non-metastatic breast tumors.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Chemistry for Life Science & Jiangsu Key Laboratory of Molecular Medicine, Medical School of Nanjing University, Nanjing 210093, China.

ABSTRACT

Background and aims: A better understanding of the effects of human adipocytes on breast cancer cells may lead to the development of new treatment strategies. We explored the effects of adipocytes on the migration and invasion of breast cancer cells both in vitro and in vivo.

Methods: To study the reciprocal effects of adipocytes and cancer cells, we co-cultured human mature adipocytes and breast cancer cells in a system devoid of heterogeneous cell-cell contact. To analyze the factors that were secreted from adipocytes and that affected the invasive abilities of breast cancer cells, we detected different cytokines in various co-culture media. To study the communication of mature adipocytes and breast cancer cells in vivo, we chose 10 metastatic pathologic samples and 10 non-metastatic pathologic samples to do immunostaining.

Results: The co-culture media of human MCF-7 breast cancer cells and human mature adipocytes increased motility of MCF-7 cells. In addition, MMP-2 was remarkably up-regulated, whereas E-cadherin was down-regulated in these MCF-7 cells. Based on our co-culture medium chip results, we chose four candidate cytokines and tested their influence on metastasis individually. We found that IGFBP-2 enhanced the invasion ability of MCF-7 cells in vitro more prominently than did the other factors. In vivo, metastatic human breast tumors had higher levels of MMP-2 than did non-metastatic tumor tissue, whereas adipocytes around metastatic breast tumors had higher levels of IGFBP-2 than did adipocytes surrounding non-metastatic breast tumors.

Conclusions: IGFBP-2 secreted by mature adipocytes plays a key role in promoting the metastatic ability of MCF-7 breast cancer cells.

Show MeSH
Related in: MedlinePlus